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BIOL1024 Fundamentals of Biochemistry > DNA replication > Flashcards

DNA replication Flashcards

1
Q

3 models of how DNA is copied

A

conservative, semi-conservative, dispersive

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2
Q

Meselson and Stahl experiment - what is used

A

E.coli, Isotopes 14N and 15N

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3
Q

Meselson and Stahl experiment - method

A
  • cells transferred to medium (14N)
  • DNA isolated of 1,2,3….generations
  • separate DNA by density gradient centrifugation
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4
Q

at 0 generation

A

all DNA are 15N

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5
Q

1 generation

A

density between 14N + 15N (intermediate)

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6
Q

2 generation

A

14N(50%) and 50% intermediate

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7
Q

3 generation

A

14N (75%) and 25% intermediate

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8
Q

Density gradient centrifugation

A

heaviest sediment hit bottom faster

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9
Q

Caesium chloride density gradient centrifugation

A
  • DNA purified from bacteria and put in caesium chloride solution
  • spun in test tube for days at certain speed
  • DNA form equilibrium
  • higher conc of caesium at bottom
  • DNA separate depending on density (buoyant density)
  • sample reaches natural buoyancy in solution
  • bands exposed in UV light against the film
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10
Q

How DNA form equilibrium

A

density of DNA = density of surrounding

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11
Q

conclusion after M and S experiment - of mixture 14N and 15N

A

semi conservative or dispersive because they show mix of 14N and 15N

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12
Q

result of redo the experiment but can be done same in denatured DNA - single stranded

A

semi-conservative - get 15N and 14N

while dispersive - still mixed 15N and 14N

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13
Q

sucrose density centrifugation

A

sample at top of tube containing gradient of sucrose concentration
more concentrated at the bottom

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14
Q

replication of E.coli

A

circular double stranded genome

starts at a fixed point and is bidirectional

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15
Q

DNA replication in Eukaryotes

A

has multiple replication forks which are the multiple sites

strands separate and DNA polymerase forming daughter cells

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16
Q

what occurs to the duplex during replication in eukaryotes

A

opens up and new bases added at 3’ -end

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17
Q

how DNA is replicated - bonding of 2 nucleotides

A
  • deoxynucleotide triphosphate (DNT) bond to primar
  • -OH in primar attacks alpha phosphate of DNT
  • breaks bond and lose 2 phosphate group - hydrolysed
  • 2 phosphate separate which produces energy which drives reaction of addition of individual bases
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18
Q

Alpha phosphate position of DNT

A

first phosphate close to deoxyribose

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19
Q

Beta phosphate position in DNT

A

second phosphate close to deoxyribose

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20
Q

Gamma phosphate position in DNT

A

third phosphate to deoxyribose

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21
Q

Lagging strand

A

3’ - 5’ direction of other strand template

slightly slower than 5’ - 3’

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22
Q

Okazaki fragments - method

A
  • pulse labelling technique
  • using radioactive labelled DNA
  • pulse radioactive molecule and harvest DNA
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23
Q

Okazaki fragments - result

A

small fragments of DNA strand which can be joined together by DNA ligase

24
Q

helicase

A

opening up DNA stand by breaking hydrogen bonds forming the multiple replication forks

25
primase
adding a short strip of RNA allowing polymerase to bond
26
polymerase II
add new nucleotides at 3' -end in 5' - 3' direction
27
polymerase I
replace RNA laid down by primase
28
ligase
reform phosphodiester linkage between lagging strands to form continuous strand
29
binding proteins
stabilise DNA strand as it is separated as single stranded DNA is unstable
30
Use of DNA polymerase for DNA sequencing
extend primer bound to single stranded fragment to be sequenced
31
what other things needed for DNA sequencing
dideoxynucleoside triphosphate, normal dNTPs
32
sanger method sequencing
- add ddATP in chain which breaks the sequence at a specific point therefore have different size fragments - denature and separate products by polyacrylamid gel electrophoresis
33
how to prevent formation of small fragments
have a lower concentration of ddNTP compared to dNTP
34
Agarose
Physical gel used as horizontal submerge
35
function of agarose
separate double stranded DNA fragment
36
how to use agarose
heated and when cooled it forms a gel substance = mesh
37
method of DNA sequencing using agarose gel
DNA is added and run through the gel to positive charge | shown by adding dye - fluoresces when bound to DNA
38
polyacrylamid gel
chemical gel
39
how to use polyacrylamid
gets a chemical reaction that cross polyacrylamid creating mesh network
40
function of polyacrylamid
for small fragments | in denaturing gel at 8M urea to separate single stranded DNA fragments
41
how is the DNA in polyacrylamid visualised
by autoradiography 32P or covalent attachment of fluorescent group
42
how to read the results of DNA sequencing
smallest fragment at the positive side reading sequences 5' to 3'
43
increase in concentration of acrylamide
smaller the size of pores
44
ddNTPs in sequencing to be seen
different colours for different bases in different or same gel
45
Next generation sequencing
- fragment DNA and anneal to slide using oligonucleotide adaptors - PCR amplified to multiple copies of that DNA - using fluorescent nucleotide for sequencing - images taken after adding new nucleotide
46
what is used next generation sequencing
reversible terminator
47
how reversible terminator is used
pause, take image, add nucleotide (reverse terminate)
48
method of reversible terminator
3' - reversible blocking group | - after image is taken, blocking group is cleaved off and adds -OH
49
cluster of PCR
generate DNA | each cluster sequenced at the same time
50
method of cluster of DNA
nucleotide is added to DNA template - picture taken - block and fluorescent dye removed - repeats to next nucleotide
51
how DNA is amplified
using PCR - polymerase chain reaction
52
method of PCR
- DNA is denatured forming single strand by increasing temperature - add short primer complementary to end of sequence of interest - decrease temperature - anneal which binds to region to amplify - thermostable DNA polymerase used for DNA extension - repetition of denaturation, annealing and extension
53
thermostable DNA polymerase
Taq | able to withstand high temperature and used to extend DNA
54
Taq
originally isolated from Thermus aquaticus
55
how cluster of OCR is generated
computer algorithms detect signals and construct sequence

BIOL1024 Fundamentals of Biochemistry (25 decks)

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